Stability evaluation of candidate reference genes for real-time qPCR normalization in Rhyzopertha dominica (Coleoptera: Bostrycidae)

Abstract Rhyzopertha dominica is a serious stored grain insect pest around the world. Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely used experimental method in molecular biology for detecting the expression of target genes. As appropriate reference genes are essential for normalizing gene expression, the selection of suitable reference genes is the basis of RT-qPCR experiments. In this study, the expression profiles of 7 candidate reference genes of rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA were analyzed under 4 different experimental conditions. The expression stability of candidate genes was evaluated using the ΔCt, GeNorm, BestKeeper, NormFinder, and RefFinder methods. The results revealed that different reference genes were suitable for various experiments. Specifically, rps3 and rps6 were appropriate for the developmental stages and all samples: 18S rRNA and rps13 for temperature-related experiments, actin and rps6 for sex-related experiments, and rps6 and gadph for starvation stress. Our results lay essential groundwork for the normalization of RT-qPCR analyses and contribute to genomic and gene functional research of R. dominica.